Determination of albumin in liquids



United States Patent 3,063,812 DETERMINATEUN 0F ALBUMEW IN LEQUIDS Galen F. (Iollins, Ellrhart, Ind, assignor to Miles Laboratories, Inc., Eiiriiart, Ind, a corporation of lndiana No Drawing. Filed Apr. 2, 1957, Ser. No. 650,068 17 (Ilaims. ((11. 23-230) This invention relates to diagnostic compositions and, more particularly, to new and improved compositions for the detection and quantitative estimation of albumin in aqueous solutions such as urine.

it is an object of the invention to provide improved diagnostic compositions in convenient tablet form which may be used with confidence, even by unskilled persons, to detect and give a reliable quantitative estimation of albumin in urine and in other aqueous solutions.

A further object is to provide diagnostic compositions for the determination of albumin in aqueous liquids which may be easily prepared from readily available materials which is capable of detecting as little as about 5 mg. percent (50 parts per million) of albumin in urine, and which may be stored over long periods of time without losing its sensitivity in testing for albumin.

Other objects and advantages of the invention will be apparent from the following description thereof.

The basis of the present invention is the phenomenon of protein error exhibited by certain indicators whereby in solutions containing protein such indicators undergo their characteristic acid-to-base color change at a lower pH value than that at which they would change color in the absence of protein. That is to say, an indicator which exhibits protein error will, in solution containing protein, under certain conditions show its basic color although the pH of the solution may actually be well below that at which the color chan e normally occurs, and the extent to which the color of the solution is changed is an indication of the amount of protein in the solution. The mechanism by which the presence of protein enables certain dyes to exhibit their basic color in solutions having a pH below the acid-to-base color change point is not clearly understood but it is known that protein is limited in its range and capacity for bringing about this effect. Specifically, this color-change of the indicator, due to the presence of protein, may be eifected only if the solution being tested has a final pH not substantially lower than about 1.25 pH units below the pH at which the normal color change of the indicator occurs.

in accordance with the invention, a diagnostic composition for determining albumin in liquids comprises a mixture of a major proportion of a solid acid reacting substance and a minor proportion of an indicator dye which exhibits protein error, the mixture being dispersed throughout an inert carrier or matrix of finely divided cellulose, and all of these ingredients being compressed into tablets of convenient size, e.g-., A in diameter and weighing about .30 gram. As will be pointed out in greater detail hereinafter, the test for albumin using such a tablet requires only that a drop of the liquid specimen to be tested, e.g.,- urine, be placed on the tablet to produce any color reaction due to the presence of albumin. If albumin is not present, the color of the tablet remains the acid color of the indicator. However, if albumin is present in the specimen it will be adsorbed by thecellulose and remain on the surface of the tablet where .it contacts the dye which is present there and causes the dye to change color while the remaining liquid portion of the specimen is absorbed by the tablet.

As would be expected, the proportion of dye in the present compositions is very small, good results being obtained using between about .02% and about .3% by weight of dye based on the weight of tablet. This range of proportions is not critical, however, and greater or 3,%3,8l2 Patented Nov. 13, 1962 '2 smaller proportions of dye may be used satisfactorily although with the smaller proportions the color is weakened rendering the test correspondingly less sensitive, and with greater proportions the excess of dye is for the most part wasted.

Inasmuch as the function of the cellulose in the compositions is to adsorb albumin on the surface of the tablet where it may be readily detected during the test, I prefer to use cellulose in such an amount that it accounts for at least 35% of the weight of the total composition. In general, I prefer the cellulose component of my compositions to comprise from about 45% to about 55% by weight of the tablet; however, greater or lower percentages of cellulose powder may be used if desired, compositions having as little as about 25% and as much as about by weight of cellulose based on the weight of the total composition giving satisfactory results. Four indicators which I have found to work very well in my improved compositions and which are preferred are brom phenol blue and brom cresol green, tetra brom phenol blue and tetrabromphenolphthalein ethyl ester. Another indicator which will react as desired, but which gives a weak color during the test, is m(p-anilinophenylazo) benzene sulfonic acid sodium salt.

The function of the acid reacting substance is to lower the pH of the urine below the acid-to-basic color change point of the indicator, which for brom phenol blue is pH 3.0, for brom cresol green is pH 3.8, for tetra brom phenol blue is pH 3.0, and for tetrabromphenolphthalein ethyl ester is pH 4.0. When urine is the liquid tested, the acid forms a buffer with the alkalinity of the urine which assists in maintaining the pH of the liquid below the color change point of the dye.

Many solid acid reacting substances, including a large group of organic acids and a number of inorganic acidreacting salts may be employed for this purpose, those having an ionization constant between about 10- and about 10 preferably about lO and which are either slightly soluble or slowly soluble in' water being preferred. Suitable acid reacting substances are citric, tartaric, giycollie, malic, fuma'ri'c, phthalic, malonic, ma'ndeli'c, glutaric, aconitic, maleic, benzoic, adipic, salicylic, gallic and gentisic acids, andaluminum sulfate and aluminum chloride. Of these I prefer to use salicylic, fumaric or phthalic acid.

The acid used must not be too strong, i.e., it must not have an ionization constant greater than 10" since such an acid would tend to rapidly reduce the pH of the albumin causing a rapid disappearance of the basic color of the dye characteristic of the positive test.

The cellulose powder used in my compositions may be manufactured from different source materials such as wood, cotton, etc. I have found the following specific powdered cellulose products satisfactory for my purposes: B quality coarse grade cellulose powder, B quality standand grade cellulose powder (both products manufactured by W 82R BalstonLiLtdJ, and alpha cellulose (a product manufactured by Brown Company and sold under the trade name of Solka Floc BW-lOO'). The products do not necessarily need to be white'ghoweven'the lighter the color, the easier it' is to observe the color changes;

Salicylic .acidu 600 Brom phenol blue 1 Powdered woodcellulose 500 Corn starch 26 tableting. If desired, other protein-free binders such as sucrose and dextrose may be used instead of the corn starch.

Example 2 The following ingredients in the proportions given, tableted as in Example 1, provide a modification of the compositions of my invention:

Parts by weight Sodium citrate-2H O 70 Citric acid anhydrous 180 Tetrabromphenolphthalein ethyl ester 0.1 Cellulose powder (whatman B quality) 300 Sucrose 26 In testing a liquid specimen for albumin one drop of the specimen is placed on a tablet prepared as described above. After this drop is absorbed, two drops of Water are placed on the tablet. If albumin is present in the specimen it is adsorbed on the surface of the tablet while the liquid (e.g., urine) soaks into the mass of the tablet. The two added drops of water wash all but the albumin and reacted dye from the surface of the tablet so that any color change due to the presence of albumin stands out in detail. The presence of albumin is indicated by the basic color of the indicator being observed on the surface of the tablet. In the absence of albumin, the color of the tablet does not change from the acidic color of the dye.

I claim:

1. A diagnostic composition comprising a mixture of a major proportion of a solid organic acid and a minor proportion of an indicator dye which exhibits protein error, said mixture being distributed throughout a matrix of powdered cellulose compressed in tablet form.

2. A diagnostic composition for determining albumin in liquids comprising a mixture of a major proportion of an acid-reacting substance selected from the group consisting of solid organic acids and acidic salts and a minor proportion of an indicator dye which exhibits protein error, and a carrier for said mixture consisting essentially of finely divided cellulose, said ingredients being intimately mixed together and compressed in tablet form.

3. A diagnostic composition in accordance with claim 2 including a protein-free binder for said ingredients selected from the group consisting of corn starch, sucrose and dextrose. r

4. A diagnostic composition for determining albumin in liquids comprising a mixture of a major proportion of a solid organic acid having an ionization constant between about 10* and about and a minor proportion of an indicator dye which exhibits protein error, and a carrier for mixture consisting essentially of finely divided cellulose, said ingredients being intimately mixed together and compressed in tablet form.

5. A diagnostic composition comprising an intimate mixture, compressed in tablet form, of a solid acid reacting substance having an ionization constant between about 10- and about 10- an indicator dye which exhibits protein error, and powdered cellulose, the dye being present in an'arnount between about .02% and about 3% by weight, said powdered cellulose being present in an amount between about 25% and about 75% by weight based on the weight of the total composition.

56. A diagnostic composition comprising an intimate mixture compressed in tablet form, of a solid acid reacting substance having an ionization constant between about 10- and about 10*, an indicator dye which exhibits proteinerror and powdered cellulose, the dye being present in an amount between about .02% and about 3% by weight, said powdered cellulose forming at least 25% of the weight of said composition.

7. A diagnostic composition for determining albumin in liquids comprising a mixture of a major proportion of a member of the group consisting of salicylic, fumaric and phthalic acids and a minor proportion of an indicator dye selected from the group consisting of brom phenol blue, brom cresol green, tetra brom phenol blue, tetrabromphenolphthalein ethyl ester and m(p-anilinophenylazo) benzene sulfonic acid sodium salt, and a carrier for said mixture consisting essentially of finely divided cellulose, said ingredients being intimately mixed together and compressed in tablet form.

8. A composition according to claim 7 wherein said indicator is brom phenol blue.

9. A composition according to claim 7 wherein said indicator is brom cresol green.

10. A composition according to claim 7 wherein said indicator is tetra brom phenol blue.

11. A composition according to claim 7 wherein said indicator is tetrabromophenolphthalein ethyl ester.

12. A diagnostic composition for determining albumin in liquids comprising a mixture of a major proportion of salicylic acid and a minor proportion of brom phenol blue, and a carrier for said mixture consisting essentially of finely divided cellulose, said ingredients being intimately mixed together and compressed in tablet form.

13. A diagnostic composition for determining albumin in liquids comprising about 600 parts by weight of salicylic acid, about one part by weight of brom phenol blue, about 560 parts by Weight of cellulose powder and about 26 parts by weight of corn starch, said ingredients being intimately mixed together and compressed in tablet form.

14. A diagnostic composition for determining albumin in liquids comprising about 70 parts by weight of sodium citrate dihydrate, about 180 parts by weight of anhydrous citric acid, about 0.1 part by weight of tetrabromphenolphthalein ethyl ester, about 300 parts by weight of cellulose powder and about 26 parts by weight of sucrose, said ingredients being intimately mixed together and compressed in tablet form.

l5. In a method of determining the presence of albumin in a liquid wherein an acid reacting substance and an indicator dye exhibiting protein error serve as the determining media, the improvement which comprises adsorbing the albumin contained in the liquid on cellulose in the presence of said determining media, and separating the non-albumin constituents of the liquid from the adsorbed albumin by washing the latter.

16. The method of determining the presence of albumin in a liquid comprising placing a drop of the liquid 'on a tablet containing in intimate admixture: a major :protein error, and a carrier proportion of an acid-reacting substance selected from the group consisting of solid organic acids and acidic salts, a minor proportion of an indicator dye which exhibits for said acid-reacting substance and dye consisting of finely divided cellulose; and,

'after said drop is absorbed in said tablet, placing thereon two drops of water, the presence of albumin being indicated by the display of the basic color of the indicator on the surface of said tablet.

17. The method of determining the presence of albumin in a liquid comprising adsorbing the albumin from a drop of said liquid on a quantity of compressed powdered cellulose in the presence of a protein determining medium consisting essentially of (a) a major proportion of an acid-reacting substance selected from the group consisting of solid organic acids and acidic salts, and (b) a minor proportion of an indicator dye which exhibits protein error, and separating the non-albumin constituents of the liquid from the adsorbed albumin by washing the latter with two drops of water, the presence of albumin being indicated by the display of the basic color of the indicator on the surface of the cellulose.

(References on following page) UNITED STATES PATENTS Fortune Sept. 5, 1939 Acree Nov. 7, 1939 Galat May 5, 1942 Goodale Mar. 23, 1943 Sheftel Oct. 12, 1943 Compton Oct. 23, 1945 Free et a1 Sept. 30, 1958 Free Nov. 10, 1 959 6 OTHER REFERENCES Abstracts of Papers, 131st Meeting Am. Chem. Soc., March 25, 1957, pages 750 and 76C.

Fiegl: Microchim. Acta, v01. 2, pages 107-110, 1937.

Kolmer et aL: Approved Lab Technic, 5th Ed. 1951, page 142.

Free: Gastroenterology, v01. 24, No. 3, July 3, 1953, pages 414 et seq. 

16. THE METHOD OF DETERMINING THE PRESENCE OF ALBUMIN IN A LIQUID COMPRISING PLACING A DROP OF THE LIQUID ON A TABLET CONTAINING IN INTIMATE ADMIXTURE: A MAJOR PROPORTION OF AN ACID-REACTING SUBSTANCE SELECTED FROM THE GROUP CONSISTING OF SOLID ORGANIC ACIDS AND ACIDIC SALTS, A MINOR PROPORTION OF AN INDICATOR DYE WHICH EXHIBITS "PROTEIN ERROR", AND A CARRIER FOR SAID ACID-REACTING SUBSTANCE AND DYE CONSISTING OF FINELY DIVIDED CELLULOSE; AND AFTER SAID DROP IS ABSORBED IN SAID TABLET, PLACING THEREON TWO, DROPS OF WATER, THE PRESENCE OF ALBUMIN BEING INDICATED BY THE DISPLAY OF THE BASIC COLOR OF THE INDICATOR ON THE SURFACE OF SAID TABLET. 